兔心室肌细胞水肿性氯电流的功能特点和分子基础

Functional characteristics and molecular basis of swelling-activated chloride conductance in the rabbit heart

目的探讨兔心室肌细胞水肿性氯电流(ICl,Swell)的分子基础及其功能特点。方法应用RT-PCR、亚克隆、DNA测序和Western印迹等方法,检测兔心室肌细胞可能携带ICl,Swell的氯离子通道亚型2、3和4(ClC-2、3、4),并用膜电钳方法检测ICl,Swell的电生理学特点。结果ClC-2为兔心室肌细胞ClC-2,ClC-3与人有97%的同源性。Western印迹分析证实了ClC-2和ClC-4的表达。ICl,Swell的外向电流与ClC-3携带的外向整流性电流特点一致;ICl,Swell的内向电流与ClC-2携带的内向整流性电流特点一致。结论ClC-2与ICl,Swell的内向整流电流有关,ClC-3与其外向整流电流有关。因抑制ICl,Swell的外向电流可阻断缺血预适应,故ClC-3可能是防止心肌缺血的分子靶目标之一。

Objective： To investigate the functional characteristics and molecular basis of the swellingactivated chloride conductance （ICl,Swell） in the rabbit heart. Methods： Candidate chloride channel subtypes （ClC-2, ClC-3 and ClC-4） in rabbit heart ventricle were determined by using RT-PCR and Western blot analysis. Whole cell ICl,Swell was recorded from isolated rabbit ventrieular myoeytes. The inhibitory effects of chloride channel blocker DIDS, NPPB and IAA-94 on ICl,Swell were examined. Results, Using PCR primers, we obtained the expected size of PCR products for ClC-2, ClC-3 and ClC-4. ClC-2 and ClC-3 expression was confirmed by automated fluorescent DNA sequencing. Western blot results showed that ClC-4 was expressed in abundance and ClC-2 was expressed at somewhat lower levels. The biological and pharmacological properties of the outward part of ICl, Swell were identical to those known properties of ClC-3 in exogenously expressed systems. Conclusion： ClC-3 might be responsible for the outwardly rectifying part of ICl,Swell and may be the molecular targets of cardioprotection associated with ischemic preconditioning or hypo-osmotic shock.